The present invention is concerned with methods for the quantitative chemical analysis of bodily fluids for total billrubin and direct reacting bilirubin content, and reagents for use in the methods.
Bilirubin is an orange-colored or yellowish substance found in bodily fluids such as blood serum, plasma and amniotic fluid. It is formed as a product of the catabolism of hemoglobin. Bilirubin is transported througout the body in blood serum attached to serum albumin. Bilirubin is conjugated with glucuronic acid in the liver to form the diglucuronide. In its water soluble conjugated glucuronide form, bilirubin enters the biliary system for excretion in the bile.
There are two forms of bilirubin found in blood, namely conjugated bilirubin and unconjugated bilirubin. Conjugated bilirubin is water soluble, whereas unconijuated biliribin is non-water soluble. "Total bilirubin" refers to the total amount of both conjugated and unconjugated bilirubin in a sample. "Direct bilirubin" or "direct reacting bilirubin" refers to the amount of conjugated bilirubin in a sample.
Generally, in healthy individuals, only low levels of bilirubin are present in bodily fluids. The nonrmal range of total bilirubin in healthy adults is about 0.2-1.0 mg/dL, with about 0.0-0.2 mg/dL conjugated bilirubin and the rest unconjugated bilirubin. Elevated serum bilirubin levels are observed in a variety of disease conditions including hernolytic disorders, biliary obstruction, cholestasis, hepatitis, cirrhosis and decreased conjugation. A condition known as bilirubinemnia is frequently a direct result of these disturbances. The quantification of amount and type of bilirubini in blood serum or plasmna is useful in diagnosing these conditions, and, following diagnosis, in monitoring treatment of the disease.
All newborns have serum unconjugated bilirubin levels greater than the normal levels found in the healthy adult population, and about 50% of newborn babies are clinically jaundiced during the first 5 days of life. In the normal full-term neonate, unconjutgated bilirubin values rise to 4 to 5 mg/dL (68-85 .mu.mol/L), and in a small percentage of newborns, these levels may rise to as high as 10 mg/dL (170 .mu.mol/L) 48 hours after birth, with a decrease to normal values in about 7 to 10 days. This temporary increase is caused by increased bilirubin production as a result of hemolysis of erythrocytes, and by incomplete bilirubin metabolism and excretion. In about 5% of neonates, unconjugated bilirubin values of greater than 15 mg/dL (256 .mu.mol/L) are seen. The increased production of bilirubin that accompanies the premature breakdown of erythrocytes and ineffective erythropoiesis results in hyperbilirubinemia in the absence of any liver abnormality.
The inherited disorders known as Gilbert's and Crigler-Najjar syndrome are known causes of hyperbilirubinemia. Since bilirubin cannot be conjugated at the normal rate in patients with these disorders, the rate of excretion of bilirubin is significantly reduced, and the serum concentration of unconjugated bilirubin increases. Serum total bilirubin concentrations of 20 to 50 mg/dL (342 to 855 .mu.mol/L) are commonly seen in patients afflicted with Crigler-Najjar syndrome Type I. Patients with this disease may the in infancy owing to the development of kernicterus (bilirubin staining of the basal ganglia of the brain). In patients with Crigler-Najjar syndrome Type II, total bilirubin concentration is less than 20 mg/dL (342 .mu.mol/L). Total bilirubin concentrations of less than 3 mg/dL (51 .mu.mol/L) are usual in individuals with Gilbert's syndrome.
In addition, in liepatobiliary diseases of various causes, bilirubin uptake, storage, and excretion are impaired to varying degrees. Both conjutated and unconjugated bilirubin are retained in these disorders, and a wide range of abnormal serum concentrations of each form of biliubin may be observed. When any portion of the biliary tree becomes blocked or subnormally permeable, biliary passage of bilirubin and of all other constituents of bile is retarded. Thus, these substances are retained. As a result, plasma concentrations of conjugated bilirubin increase to abnormal values.
Total bilirubin assay must measure both the water soluble conjugated and the non-water soluble unconjugated bilirubin. Direct reacting bilirubin measurement is targeted to principally measure conjugated bilirubin. Thus, when total bilirubin and direct reacting bilirubin are measured, the relative amount of uncon jugated bilirubin is readily indicated by subtracting the direct reacting biliubin from the total bilirubin.
In many cases it is very important to distinguish between conjugated (direct) bilirubin and unconjugated bilirubin because the diagnostic implications of each are different. For example, in Rh disease of newborns, it is known that levels of about 20 mg/dL of unconjugated bilirubin in serum places the infant at risk of death or permanent brain damage due to the development of kernicterus. Treatment for high unconjugated bilirubin levels involve exchange blood transfusions which may themselves be life threatening and may place the infant at risk of contracting AIDS or other viral diseases. However, similar levels of conjugated bilirubin may be tolerated by the infant with no lasting effects. It is therefore important to be able to distinguish the type of bilirubin for both diagnosis and treatment.
Most bilirubin assays have been based on the diazo reaction reported by Ehrlich in 1883. A later diazo method for total bilirubin assay originally was reported by Jendrassik and Grof in 1938, and later modified by Doumas et al. in 1973. This modified Jendrassik and Grof method may be considered as the reference method by which new methods are compared. This method employs an azosulfanilic acid prepared by combining sodium nitrite with sulfanilic acid. Direct reacting bilirubin assays have used diazo reactions of various types under acid conditions in order to achieve relative specificity for conjugated bilirubin in the presence of unconjugated bilirubin. Under these reaction conditions, most of the "direct bilirubin" detected in the reaction is conjugated bilirubin. Although some of the color may be due to unconjugated bilirubin, the small amount of unconjugated bilirubin does not significantly reduce the diagnostic value of the test for detecting direct bilirubin.
Since most total and direct bilirubin assays are performed in clinical laboratories losing automated biochemical analyzers, the modified Jendrassik and Grof method is not widely used for these assays due to its poor suitability for automation. For example, the modified Jendrassik and Grof method requires the use of as many as four separate reagents while the most commonly used analyzers are designed to use only two reagents. Additionally, the azosulfanilic acid reagent used in the Jendrassik and Grof method is stable for only about one day and must be prepared regularly by the laboratory. Further, the ascorbic acid utilized in the reaction is unstable and must be prepared as often as daily.
Attempts have been made to improve the Jendrassik and Grof method. In U.S. Pat. No. 3,569,721, Denney et al. attempted to improve the Jendrassik and Grof method by replacing the ascorbic acid constituent with stable hydroxylamnine salts. Although this method avoided the use of the unstable ascorbic acid, it was still necessary to use the unstable azostilfanilic acid reagent. The method also required the use of four separate reagents. Thus, this method was unsuitable for most currently used clinical laboratory automated analyzers.
A number of other modifications of the diazo reaction have been made to simplify the reaction for automated use. Most of these attempts have used fast diazonium salts, such as 2,4-dichlorophenyl or 2,5-dichlorophieniyl diazonium salts as diazo reagents, such as the methods described in U.S. Pat. Nos. 3,754,862 and 3,754,862. However, R. Poon. et al, Clin. Chem. 31, 92-94, 1985, describe severe interference in this type of diazo method due to the presence of indican in the serum of patients with renal failure. The interference from indican invalidates the results of the bilirubin test using these prior art methods for the analysis of the serum of such patients. This represents a serious problem in the use of these methods.
The total bilirubin level in bodily fluids of infants has been measured by direct spectrophotometric estimation at 454 nm with correction at 540 nm However, this method cannot be applied to older children or adults due to the presence of carotene and other pigments in the serum. See, Tietz, N., Textbook of Clinical Chemistry, W.B. Saunders Company, (198(p. 1386. A device enmbo(lying direct spectrophotometric observation has been described in Goldberg, U.S. Pat. No. 3,569,721.
The specificity of the measuremnent of adult bilirubin by direct spectrophotometric observation has been improved by making observations before and after the destriction of bilirubin with the enzyme bilirubin oxidase. Although the enzyme treatment improves specificity, the enzyme reagents are unstable and do not solve the stability problems of the diazo methods. Additionally, enzymes are costly, relative to diazo reagents.
In Ann. Clin. Biochem., 30:175-179, (1993), O'Leary et al described a two-step method based upon the measurement of the absorbance of bilirubin itself at about 480 nm, followed by measurement of the absorbance at 480 nm after destruction of the bilirubin with ferricyanide. The reaction was carried out at neutral pH conditions. Although the O'Leary methods, represented an improvement in accuracy and stability in comparison to diazo methods, the method is limited due to the use of the 460-480 nm wavelengths for detection. At these wavelengths, highly lipemic or hemolyzed sera may cause the absorbance of the sample plus bilirubin to exceed the detection capability of automated systems, and therefore, a false result may be produced. Additionally, proper mixing of the first reagent with the serum is critical prior to the measurement of the absorbance at 480 nm in the first step. Automated devices do not always provide the ability to mix a first reagent and serum prior to the addition of a second reagent. When the method is applied to such systems, the precision is not satisfactory for clinical use.
In 1964 Ferro and Ham (U.S. Pat. No. 3,348,920), taught that total bilirubin could be quantified by reacting the bilirubin in a supernatant solution under acid conditions and in the presence of ferric ion. Prior to this reaction, the protein in the sample is precipitated and removed from the solution. This method requires multiple steps and a total time of tip to thirty minutes. The time and the multiple steps required in this method are incompatible with the types of automated analyzers commonly used in clinical laboratories, in particular, due to the necessity of performing filtration or centrifugation for separation of the precipitate. Additionally, the reactants in the Ferro/Ham method include highl levels of iron. Iron is a potential contaminant that may adversely affect the results of other assays, such as serum iron, that are frequently performed on the same automated equipment. Ferro and Ham did not teach a method for direct bilirubin assay, as the method detects only total (both unconjugated and conjugated) bilirubin.
Serious errors in most, if not all, commonly used total and direct bilirubin assays are caused by the presence of lipemia (fatty substances) and hemolysis (red blood cell contents) in the sample to be assayed. Glick has reported that a significant percentage of blood samples presented to clinical laboratories for assay contain these interferences. M. Glick, Interferographs: User's Guide to Interferences in Clinical Chemistry Instruments, Second Edition, 1991, Science Enterprises, Inc. Indianapolis, Ind. Glick et al. have reviewed the performance of most commonly used clinical chemistry automation in the presence of lipemia and hemolysis in the sample. Melvin R. Glick and Kenneth W. Ryder, Clinical Chemistry 33 pages 1453-1458, 1987. The work of Glick indicates that bilirubin assays are among the most prone to interference from lipemia and hemolysis of all commonly performed diagnostic assays.
Thus, prior art automated methods continue to be subject to serious errors. In addition, the reagents used in such prior art methods often have limited stability. Accordingly, it is desired to provide a method for assay of total and direct reacting bilirubin in both adult and infant bodily fluid samples that overcomes the problems and limitations encountered with prior art methods, and that utilizes reagents that are suitable for use with automated laboratory analyzers.